Project Summary/Abstract Tumor resistance to radiotherapy continues to be a significant problem in improving outcomes of patients diagnosed with locally advanced, unresectable cancers including head and neck (HNC). Cellular sensitivity to ionizing radiation (IR) is governed by intracellular and extracellular factors. To help overcome tumor radioresistance, drugs that sensitize tumor to ionizing radiation (IR) are used. In theory, more potent radiosensitizers should increase tumor kill and improve patient outcomes. In practice, the clinical utility of of such drugs is curtailed by side effects that limit doses. To address these issues, we are developing activatable cell penetrating peptide (ACPP) probes to monitor and selectively deliver conjugated radiosensitizers to tumors based on extracellular tumor protease activity. ACPP consist of a drug conjugated polycationic cell penetrating peptide and an autoinhibitory polyanionic peptide separated from each other by a flexible peptide linker. Our initial peptide linker is cleaved by matrix metalloproteinases (MMP) 2/9, abundantly expressed in tumors. Intact, drug conjugated ACPP is ?pro-drug? where the the drug linked cell penetrating peptide is neutralized by the polyanionic peptide, blocking intracellular drug uptake. Upon encountering MMP 2/9 within tumors, ACPP is cleaved (i.e. ?activated?) and releases the drug conjugated cell-penetrating peptide, which is then taken up by tumor cells. An advantage of our ACPP probes is that they can be designed to non-invasively image extracellular tumor protease activities by synthesizing ratiometric ACPP. Ratiometric ACPP are labeled with Cy5 and Cy7 on the polycationic and polyanionic peptide respectively, acting as a FRET donor/acceptor pair. By using different protease sensitive peptide linker sequences between the two charged polypeptides, ratiometric ACPP Cy5:Cy7 emission ratio non-invasively measures distinct tumor protease activities in situ. A unique property of ACPP is the dependency on protease cleavage begets enzymatic amplification in the area of interest. The goals of our proposal are to develop ACPP probes to selectively radiosensitize tumors. In Aim 1, we will test the pharmacokinetics, biodistribution and efficacy of ACPP conjugated radiosensitizers in murine HNC models. To achieve this, drugs and fluorophores will be conjugated to ACPP and combined with IR. In Aim 2, we will interrogate in situ how IR alters extracellular protease activities in a dose dependent and temporal manner using a panel of ratiometric ACPP with distinct linkers cleaved preferentially by specific proteases. Identifying IR inducible ACPP cleavage will promote IR guided drug delivery development. In summary, ACPP radiosensitizer delivery has several innovative features that warrant pre-clinical inquiry. Diagnostically, ratiometric ACPP allows for non-invasive imaging of intrinsic tumor microenvironment proteases and IR's extrinsic influence. Therapeutically, ACPP's modular architecture allows for permutations of different classes radiosensitizing drugs to be conjugated to ACPPs with distinct protease linkers. Together, our studies will test how ACPPs can be exploited to improve the therapeutic ratio of radiotherapy.